We propose to elucidate the structural architecture and the catalytic mechanisms of the mitochondrial machinery responsible for the synthesis of iron-sulfur clusters (ISC). ISC are highly versatile co-factors, and cells utilize ISC-containing enzymes in a variety of capacities in the mitochondria, cytoplasm, and the nucleus. Yeast and animal cells carry out ISC synthesis primarily in the mitochondrial matrix. In addition, mitochondrial ISC synthesis is a key regulator of cellular iron uptake and iron distribution within the cell. Defects in mitochondrial ISC synthesis result in severe mitochondrial deficits (altered energy Significance metabolism, mitochondrial iron overload, iron-dependent oxidative damage, loss of mitochondrial DNA integrity), with concomitant extra-mitochondrial deficits (e.g. nuclear genome instability). In humans, such defects lead to neurodegenerative disease, and are probably implicated in age-dependent intracellular iron accumulation and age-dependent genome instability. Our limited mechanistic understanding of this process hampers our ability to interrogate its functionality directly in normal conditions, aging, and age-related disease states. Several conserved proteins are known to participate in mitochondrial ISC synthesis but it is virtually unknown how they function together. We hypothesize that ISC synthesis is a multi-step process that requires stable contacts among the proteins that catalyze the individual steps. Our rationale is that multi-component complexes can enable the protected delivery of potentially toxic iron and Innovation sulfur to scaffold proteins, as well as the protected transfer of oxygen-labile clusters from scaffold proteins to apo-enzymes. Our preliminary studies suggest that in yeast and human mitochondria ISC synthesis occurs on stable complexes made of multiple copies of three core components (iron-donor, sulfur-donor, and scaffold) reaching molecular masses of megadaltons. The identity of additional components, protein- protein interaction surfaces, catalytic mechanisms and overall architecture of these macromolecular machines are the focus of our specific aims: Aim 1: Define the protein composition of native ISC biosynthetic machineries and their sub-complexes isolated from yeast and human cells; Aim 2: Reconstitute enzymatically active sub-complexes and whole machineries in vitro and in tractable cellular systems; Aim 3: Approach Define the structures of sub-complexes and the whole machinery. This plan teams-up biochemists and molecular biologists with protein structural biologists, making optimal use of their complementary expertise and resources. Our research will elucidate the details of a fundamental process that remains poorly understood mechanistically. It will also provide new information and tools needed to identify and better understand human deficits in ISC synthesis and to develop approaches to modulate this process pharmacologically. Impact